Biomedical subjects
Publications and source records attributed to Ronald L Calabrese.
Researchers working on neuronal networks are increasingly taking advantage of the ability to overexpress particular molecular components such as channels. What hope do such studies hold for understanding neuronal phenotype and its role in network function?
We have designed, fabricated, and tested an analog integrated-circuit architecture to implement the conductance-based dynamics that model the electrical activity of neurons. The dynamics of this architecture are in accordance with the Hodgkin-Huxley formalism, a widely exploited, biophysically plausible model of the dynamics of living neurons. Furthermore the architecture is modular and compact in size so that we can implement networks of silicon neurons, each of desired complexity, on a single integrated circuit. We present in this paper a six-conductance silicon-neuron implementation, and characterize it in relation to the Hodgkin-Huxley formalism. This silicon neuron incorporates both fast and slow ionic conductances, which are required to model complex oscillatory behaviors (spiking, bursting, subthreshold oscillations).
To address the general problem of intersegmental coordination of oscillatory neuronal networks, we have studied the leech heartbeat central pattern generator. The core of this pattern generator is a timing network that consists of two segmental oscillators, each of which comprises two identified, reciprocally inhibitory oscillator interneurons. Intersegmental coordination between the segmental oscillators is mediated by synaptic interactions between the oscillator interneurons and identified coordinating interneurons. The small number of neurons (8) and the distributed structure of the timing network have made the experimental analysis of the segmental oscillators as discrete, independent units possible. On the basis of this experimental work, we have made conductance-based models to explore how intersegmental phase and cycle period are determined. We show that although a previous simple model, which ignored many details of the living system, replicated some essential features of the living system, the incorporation of specific cellular and network properties is necessary to capture the behavior of the system seen under different experimental conditions. For example, spike frequency adaptation in the coordinating interneurons and details of asymmetries in intersegmental connectivity are necessary for replicating driving experiments in which one segmental oscillator was injected with periodic current pulses to entrain the activity of the entire network. Nevertheless, the basic mechanisms of phase and period control demonstrated here appear to be very general and could be used by other networks that produce coordinated segmental motor outflow.
Two tubular hearts propel blood through the closed circulatory system of the medicinal leech. The hearts are myogenic but are driven by a centrally generated motor pattern that controls heart rate and intersegmental coordination. In two consecutive papers, we address the question of how the motor pattern is translated into the pattern of diastole and systole of leech hearts. We imaged the constriction patterns of the hearts in quiescent intact animals. In one heart, systole progresses rear-to-front (peristaltic coordination mode), whereas systole occurs nearly simultaneously in the other heart (synchronous coordination mode) with regular switches between these two coordination modes. Intersegmental phase relations between heart segments do not vary with changes in the heartbeat period. The peristaltic heart drives blood forward through itself and then rearward through the other longitudinal vessels. The synchronous heart does not seem to contribute to rearward flow along the body axis and may support segmental circulation instead. Simultaneous monitoring of heart motor neuron discharge and the constriction of the corresponding heart segment in innervated, reduced preparations enabled us later to meld the constriction pattern with the fictive motor pattern described in the following paper. Current injections into one heart modulatory neuron while monitoring intravascular pressure from the corresponding heart showed that these neurons can acutely change diastolic and systolic pressure. However, they do not determine the different systolic pressure profiles associated with the two coordination modes, which appear to result from the constriction pattern.
The rhythmic beating of the tube-like hearts in the medicinal leech is driven and coordinated by rhythmic activity in segmental heart motor neurons. The motor neurons are controlled by rhythmic inhibitory input from a network of heart interneurons that compose the heartbeat central pattern generator. In the preceding paper, we described the constriction pattern of the hearts in quiescent intact animals and showed that one heart constricts in a rear-to-front wave (peristaltic coordination mode), while the other heart constricts in near unison over its length (synchronous coordination mode) and that they regularly switch coordination modes. Here we analyze intersegmental and side-to-side-coordination of the fictive motor pattern for heartbeat in denervated nerve cords. We show that the intersegmental phase relations among heart motor neurons in both coordination modes are independent of heartbeat period. This finding enables us to combine data from different experiments to form a detailed analysis of the relative phases, duty cycle, and intraburst spike frequency of the bursts of the segmental heart motor neurons. The fictive motor pattern and the constriction pattern seen in intact leeches closely match in their intersegmental and side-to-side coordination, indicating that sensory feedback is not necessary for properly phased intersegmental coordination. Moreover, the regular switches in coordination mode of the fictive motor pattern mimic those seen in intact animals indicating that these switches likely arise by a central mechanism.
To survive, animals must constantly make behavioral choices. The analysis of simple, almost binary, behavioral choices in invertebrate animals with restricted nervous systems is beginning to yield insight into how neuronal networks make such decisions.
At the core of the rhythmically active leech heartbeat central pattern generator are pairs of mutually inhibitory interneurons. Synaptic transmission between these interneurons consists of spike-mediated and graded components, both of which wax and wane on a cycle-by-cycle basis. Low-threshold Ca2+ currents gate the graded component. Ca imaging experiments indicate that these low-threshold currents are widespread in the neurons and that they contribute to neuron-wide changes in internal background Ca2+ concentration (Ivanov and Calabrese, 2000). During normal rhythmic activity, background Ca2+ concentration oscillates, and thus graded synaptic transmission waxes and wanes as the neurons move from the depolarized to the inhibited phases of their activity. Here we show that in addition to gating graded transmitter release, the background Ca2+ concentration changes evoked by low-threshold Ca2+ currents modulate spike-mediated synaptic transmission. We develop stimulation paradigms to simulate the changes in baseline membrane potential that accompany rhythmic bursting. Using Ca imaging and electrophysiological measurements, we show that the strength of spike-mediated synaptic transmission follows the changes in background Ca2+ concentration that these baseline potential changes evoke and that it does not depend on previous spike activity. Moreover, we show using internal EGTA and photo-release of caged Ca2+ and caged Ca2+ chelator that changes in internal Ca2+ concentration modulate spike-mediated synaptic transmission. Thus activity-dependent changes in background Ca2+, which have been implicated in homeostatic regulation of intrinsic membrane currents and synaptic strength, may also regulate synaptic transmission in an immediate way to modulate synaptic strength cycle by cycle in rhythmically active networks.
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Rhythmic activity within the heartbeat pattern generator of the medicinal leech is based on the alternating bursting of mutually inhibitory pairs of oscillator heart interneurons (half-center oscillators). Bicuculline methiodide has been shown to block mutual inhibition between these interneurons and to cause them to spike tonically while recorded intracellularly (Schmidt and Calabrese, 1992). Using extracellular recording techniques, we show here that oscillator and premotor heart interneurons continue to burst when pharmacologically isolated with bicuculline, although the bursting is not robust in some preparations. We propose that a nonspecific leak current introduced by the intracellular microelectrode suppresses endogenous bursting activity to account for the discrepancy with results using intracellular recording. A two-parameter bifurcation diagram (E(leak) vs g(leak)) of a mathematical model of a single heart interneuron shows a narrow stripe of parameter values where bursting occurs, separating large zones of tonic spiking and silence. A similar analysis performed for a half-center oscillator model outlined a much larger area of bursting. Bursting in the half-center oscillator model is also less sensitive to variation in the maximal conductances of voltage-gated currents than in the single-neuron model. Thus, in addition to ensuring appropriate bursting characteristics such as period, phase, and duty cycles, the half-center configuration enhances oscillation robustness, making them less susceptible to random or imposed changes in membrane parameters. Endogenous bursting, in turn, ensures appropriate bursting if the strength of mutual inhibition is weakened and limits the minimum period of the half-center oscillator to a period near that of the single neuron.
We tested predictions of a computational model (Hill et al., 2002) of the leech heartbeat timing network. The timing network consists of two segmental oscillators located in the third (G3) and fourth (G4) segmental ganglia. Each oscillator consists of two reciprocally inhibitory oscillator interneurons along with the coordinating interneuron fibers that link them. In the model, the network was driven to cycle periods around the normal period of the network by repeatedly stimulating one of the paired oscillator interneurons in G3 or G4. Here we replicate these experiments in the biological system. The model predicts that the G3 and G4 oscillators can entrain the timing network to periods faster but not slower than the inherent period of the nondriven ("follower") oscillator and that they can do so symmetrically. The biological system can be driven to periods both faster (such that the driven oscillator leads in phase) and slower (such that the driven oscillator lags in phase) than the inherent period of the timing network. Although both oscillators can entrain the network, the G4 oscillator does so over a narrower range of periods. Two differences between the assumptions of the model and the properties of the biological network, spike frequency adaptation in coordinating interneurons and asymmetry in the connections from the oscillator interneurons to the coordinating interneurons, may account for these discrepancies. Individual coordinating interneurons were also able to entrain the oscillators but with little effect of the phase relationship between the oscillators, suggesting that phase relations are determined by properties inherent to the oscillator interneurons.
Motor pattern generating networks that produce segmentally distributed motor outflow are often portrayed as a series of coupled segmental oscillators that produce a regular progression (constant phase differences) in their rhythmic activity. The leech heartbeat central pattern generator is paced by a core timing network, which consists of two coupled segmental oscillators in segmental ganglia 3 and 4. The segmental oscillators comprise paired mutually inhibitory oscillator interneurons and the processes of intersegmental coordinating interneurons. As a first step in understanding the coordination of segmental motor outflow by this pattern generator, we describe the functional synaptic interactions, and activity and phase relationships of the heart interneurons of the timing network, in isolated nerve cord preparations. In the timing network, most (approximately 75%) of the coordinating interneuron action potentials were generated at a primary spike initiation site located in ganglion 4 (G4). A secondary spike initiation site in ganglion 3 (G3) became active in the absence of activity at the primary site. Generally, the secondary site was characterized by a reluctance to burst and a lower spike frequency, when compared with the primary site. Oscillator interneurons in G3 inhibited spike activity at both initiation sites, whereas oscillator interneurons in G4 inhibited spike activity only at the primary initiation site. This asymmetry in the control of spike activity in the coordinating interneurons may account for the observation that the phase of the coordinating interneurons is more tightly linked to the G3 than G4 oscillator interneurons. The cycle period of the timing network and the phase difference between the ipsilateral G3 and G4 oscillator interneurons were regular within individual preparations, but varied among preparations. This variation in phase differences observed across preparations implies that modulated intrinsic membrane and synaptic properties, rather than the pattern of synaptic connections, are instrumental in determining phase within the timing network.
We have created a computational model of the timing network that paces the heartbeat of the medicinal leech, Hirudo medicinalis. The rhythmic activity of this network originates from two segmental oscillators located in the third and fourth midbody ganglia. In the intact nerve cord, these segmental oscillators are mutually entrained to the same cycle period. Although experiments have shown that the segmental oscillators are coupled by inhibitory coordinating interneurons, the underlying mechanisms of intersegmental coordination have not yet been elucidated. To help understand this coordination, we have created a simple computational model with two variants: symmetric and asymmetric. In the symmetric model, neurons within each segmental oscillator called oscillator interneurons, inhibit the coordinating interneurons. In contrast, in the asymmetric model only the oscillator interneurons of one segmental oscillator inhibit the coordinating interneurons. In the symmetric model, when two segmental oscillators with different inherent periods are coupled, the faster one leads in phase, and the period of the coupled system is equal to the period of the faster oscillator. This behavior arises because, during each oscillation cycle, the oscillator interneurons of the faster segmental oscillator begin to burst before those of the slower oscillator, thereby terminating spike activity in the coordinating interneurons. Thus there is a brief period of time in each cycle when the oscillator interneurons of the slower segmental oscillator are relieved of inhibition from the coordinating interneurons. This "removal of synaptic inhibition" allows, within certain limits, the slower segmental oscillator to be sped to the period of the faster one. Thus the symmetric model demonstrates a plausible biophysical mechanism by which one segmental oscillator can entrain the other. In general the asymmetric model, in which only one segmental oscillator has the ability to inhibit the coordinating interneurons, behaves similarly, except only one segmental oscillator can control the period of the system. In addition, we simulated physiological experiments in which a "driving" stimulus, consisting of alternating positive and negative current steps, was used to control a single oscillator interneuron and thereby entrain the activity of the entire timing network.
Considerable experimental and theoretical effort has been exerted to understand how constant intersegmental phase relationships are produced between oscillators in segmentally organized pattern generating networks. The phase relationship between the segmental oscillators in the isolated timing network of the leech heartbeat central pattern generator is quite regular within individual preparations. However, it varies considerably among different preparations. Our goal is to determine how the phase relationships in this network are established. Here we assess whether inherent period differences, as suggested by the excitability-gradient hypothesis, play a role in establishing the phase relationships between the two coupled segmental oscillators of the leech heartbeat timing network. To do this we developed methods for reversibly uncoupling the segmental oscillators (sucrose knife) and pharmacological manipulation of the individual oscillators (split bath). Differences in inherent cycle periods between the third and fourth segmental oscillators (G3 and G4) were present in most (20 of 26) preparations. These period differences correlated with the phase differences observed between the segmental oscillators in the recoupled timing network, such that the oscillator with the faster cycle period, regardless of the segment in which it was located, led in phase in proportion to its period difference with the other oscillator. The phase differences between the original (coupled) and recoupled states of individual preparations were similar. Thus application and removal of the sucrose knife did not alter the period difference between the segmental oscillators in the timing network. Pharmacological manipulation of the uncoupled oscillators to alter the period difference between the oscillators led to similar correlated phase differences in the recoupled timing network. Across all experiments the uncoupled segmental oscillator with the faster cycle period established the cycle period of the timing network when recoupled. In conclusion, our findings indicate that an excitability-gradient plays a role in establishing the phase relationship between the segmental oscillators of the leech heartbeat central pattern generator since inherent period differences present between the oscillators are correlated to the phase relationships of the coupled/recoupled timing network.